Negrete Alejandro, Kotin Robert M
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, MD 20892, USA.
J Virol Methods. 2007 Nov;145(2):155-61. doi: 10.1016/j.jviromet.2007.05.020. Epub 2007 Jul 2.
The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g. > or =10(16) particles per production run, utilizes baculovirus expression vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred tank bioreactors are single-use, disposable bioreactors, e.g. Wave. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave bioreactors. Comparable yields of rAAV, approximately 2E+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration.
生产重组腺相关病毒（rAAV）的传统方法依赖于贴壁哺乳动物细胞的瞬时转染。为了获得认可并符合现行良好生产规范（cGMP），临床级rAAV生产工艺应具备以下特点：简单性、一致性、成本效益和可扩展性。目前，大规模生产rAAV（例如每次生产运行≥10¹⁶个颗粒）的唯一可行方法是利用杆状病毒表达载体（BEV）和昆虫细胞悬浮培养。先前描述的在40 L培养物中使用搅拌罐生物反应器生产rAAV需要特殊的实施和操作条件，并非所有实验室都具备这些条件。在搅拌罐生物反应器中生产rAAV的替代方法是一次性使用的生物反应器，例如Wave。一次性袋子在购买时已预先灭菌，从而无需最终用户进行灭菌，也避免了生产批次之间的清洁步骤，从而简化了生产过程。在本研究中，对搅拌罐生物反应器和Wave生物反应器中rAAV的生产进行了比较。搅拌罐生物反应器的工作体积为10 L和40 L，Wave生物反应器的工作体积为5 L和20 L。在所有体积和配置中均获得了相当的rAAV产量，每升细胞培养物约为2×10¹³个颗粒。这些结果表明，使用BEV大规模生产rAAV具有可重复性、可扩展性，且与生物反应器配置无关。
